Project Summary There are currently no approved vaccines for most emerging biological pathogens (Ebola, Chikungunya, MERS CoV, SARS, Zika), opportunistic infections (S. aureus, Candidiasis, adenovirus) or potential bioterrorism agents (Y. pestis, B. Pseudomallei, F. tularensis, Bunyaviridae, Flaviviridae), and therapeutic interventions such as antibiotics and antivirals are only effective for a select few pathogens. A promising approach for rapidly neutralizing the risk of pathogen exposure is the use of immunomodulators capable of eliciting a robust innate immune response within hours of administration that would provide protective resistance against a wide range of biological agents. Innate immunity has evolved as a first line of defense against invading pathogens and acts through highly conserved pattern- recognition receptors such as Toll-like receptors (TLR) to coordinate the innate inflammatory response to both endogenous and exogenous stimuli. The objective of this Phase I research proposal is to initiate an investigation of structure-activity relationships within a new class of chemically and metabolically more stable synthetic TLR4 agonists in order to identify the structural requirements for the development of a broad-spectrum immunomodulator that would provide non-specific protective resistance (NSR) against a wide range of biological agents. In addition to potentially providing weeks-long NSR against a wide range of viruses and bacteria, the improved stability profile of these synthetic immunomodulators should improve thermal stability leading to a longer shelf-life and avoidance of cold-chain requirements for distribution. Aim 1 will focus on (i) the synthesis of six new TLR4 agonists in which the labile ester and phosphate groups are replaced with more stable bioisosters, and (ii) evaluation of the stability of these new agonists in aqueous formulations. Aim 2 will evaluate the biological activity of the new compounds in vitro for (i) TLR4 species-specificity and potency in a TLR4 specific reporter assay and (ii) cytokine induction from human peripheral blood mononuclear cells. This phase I research proposal and subsequent phase II have the potential to develop broad- spectrum immunomodulators that would provide non-specific protective resistance (NSR) against a wide range of biological agents, and is primarily targeted for preventing upper respiratory tract infections in individuals or populations at risk for emerging or opportunistic pathogen exposure. Such treatment could help prevent deaths associated with seasonal or pandemic influenza viruses as well as other respiratory pathogens of significant medical concern.